A sulfur-free peptide mimic of surfactant protein B (B-YL) exhibits high in vitro and in vivo surface activities

Materials

HPLC grade chloroform, methanol, trifluoroethanol (TFE), and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA 15275), trifluoroacetic acid from Sigma Chemical Co (Saint Louis, MO 63103), NMR quality deuterated water was from Aldrich Chemical Co. (St. Louis, MO 63103), and Sephadex LH-20 chromatography gel from Pharmacia (Uppsala, Sweden). Phospholipids were supplied by Avanti Polar Lipids (Alabaster, AL 35007), and Sodium Dodecyl Sulfate (SDS) detergent was from Sigma Chemical Co (Saint Louis, MO 63103).

The Super Mini-B (SMB) peptide sequence (Figure 1A) was synthesized using a standard Fmoc protocol with a Symphony Multiple Peptide Synthesizer (Protein Technologies, Inc., Tucson, AZ 87514) or a CEM Liberty microwave synthesizer (CEM Corporation, Mathews, NC 28104), cleaved-deprotected and purified using reverse phase HPLC as described earlier^14,18. This synthesis protocol included folding of the peptide in a structure-promoting TFE-buffer solvent system to promote oxygen-mediated disulfide linkages between Cys-8 and Cys-40 and between Cys-11 and Cys-34^10,14. This covalently stabilized connectivity gave the peptide a helix-hairpin conformation, comparable to the topological organization seen for the N- and C-terminal helical domains of the saposin family of proteins^9,10,12. The synthesis of B-YL was identical to that of SMB, except for replacing cysteines with tyrosines (Tyr-8, Tyr-11, Tyr-34, and Tyr-40) and methionines with leucines (Leu-21 and Leu-28), and also omitting the oxidation step. The purified SMB and B-YL peptides were each freeze-dried directly, and the masses were confirmed by MALDI TOF mass spectrometry as described previously¹⁷. Peptide concentrations were routinely quantitated using UV absorbance based on the assay procedure developed by Anthis and Clore³¹.

Preparation of proteins and lipids in surfactant dispersions

Peptide and lipids were formulated as lipid-peptide dispersions to have a total of 3% by mole fraction of SMB or B-YL and 35 mg of total lipid [i.e., DPPC: POPC: POPG 5:3:2 mole:mole:mole] per mL of dispersion¹⁸. The peptide was dissolved in 10 mL of trifluoroethanol and co-solvated with the lipid in chloroform, followed by removal of the solvents with a stream of nitrogen gas and freeze drying of the resulting lipid-peptide film to remove residual solvent. The film was then dispersed with Phosphate Buffered Saline and the sample flask containing the hydrated film was rotated for 1 h at 60°C to produce a solution of multilamellar vesicles (MLVs)¹⁴. Lipid controls were similarly prepared but without peptide. These dispersions were then stored at 4°C prior to structural and functional measurements. To determine the molecular mass of peptides formulated with lipids, the peptide was separated from lipid using normal phase chromatography with Sephadex LH-20³². Mass spectral analysis of the B-YL peptide indicated there was no change in the molecular weight due to oxidation of tyrosines or tryptophan for one year when formulated with surfactant lipids.

Circular dichroism (CD) spectroscopy of the secondary structure of the B-YL mimic

CD spectra (190–260 nm) of the B-YL peptide in various structure-promoting environments, including surfactant dispersions, were measured with a JASCO 715 spectropolarimeter (Jasco Inc., Easton MD 21601). The instrument was routinely calibrated for wavelength and optical rotation using 10-camphorsulphonic acid³³. The sample solutions were scanned using 0.01 cm pathlength cells at a rate of 20 nm per minute, a sample interval of 1 nm, and a temperature of 37°C. Sample concentration was determined by UV absorbance at 280 nm³¹. Peptide concentration was 100 μM in sample solutions with either TFE:Phosphate buffer (10 mM, pH 7.0) having a volume ratio of 4:6 (v/v), SDS micelles (100 mM) in phosphate buffer (10 mM, pH 7.0), or Single Unilamellar Vesicles (SUVs) of simulated surfactant lipids (DPPC: POPC: POPG; 5:3:2, mole:mole:mole). Surfactant lipid SUVs were prepared at a concentration of 2.6 μM lipids/mL of phosphate buffer solution (10 mM, pH 7.0) by bath sonication for 10 minutes (https://avantilipids.com/tech-support/liposome-preparation/). Sample spectra were baseline corrected by subtracting spectra of protein-free solution from that of the protein-bound solution and expressed as the Mean Residue Ellipticity [θ] _MRE as shown in equation (1):

The symbol θ is the measured ellipticity in millidegrees, l is the pathlength in cm, N is the number of residues in the peptide, and c is the concentration of the peptide in mM.

Quantitative estimates of the secondary structural contributions were also made with SELCON 3³⁴ using the spectral basis set for membrane proteins, option 4 implemented from the DichroWeb website^35,36.

Attenuated-Total-Reflectance Fourier-transform infrared (ATR-FTIR) spectrometry of the B-YL and SMB peptides

ATR-FTIR spectra were recorded at 37°C using a Bruker Vector 22 FTIR spectrometer (Pike Technologies, Fitchburg, WI 53719) with a deuterium triglyceride sulfate (DTGS) detector. The spectra were averaged over 256 scans at a gain of 4 and a resolution of 2 cm^-114. For spectra of B-YL and SMB in TFE solutions, self-films were first prepared by air-drying peptide originally in 100% HFIP onto a 50 × 20 × 2 mm, 45° attenuated total reflectance (ATR) crystal for the Bruker spectrometer. The dried peptide self-films were then overlaid with solutions containing 40% TFE/60% deuterated-10 mM sodium phosphate buffer (pH 7.4), at a peptide concentration of 470 μM. Control solvent samples were similarly prepared for FTIR analysis, but without peptide. Spectra of peptides in solvent were obtained by subtraction of the solvent spectrum from that of peptide solvent. For FTIR spectra of B-YL and SMB in either SDS micelles or surfactant lipids, each lipid-peptide solution was transferred onto a germanium ATR crystal. The aqueous solvent was then removed by flowing nitrogen gas over the sample to produce a thick lipid-peptide (lipid:peptide ratios of 10:1, mole:mole)¹⁴. The multilayer film was then hydrated to ≥35% with deuterated water vapor in nitrogen for 1 h before acquiring the spectra³⁷. The spectra for either the B-YL or SMB peptides in the film were obtained by subtracting the spectrum of a peptide-free control sample from that of the peptide-bound sample. The relative amounts of α-helix, β-turn, β-sheet, or random (disordered) structures in lipid-peptide films were estimated using Fourier deconvolution (GRAMS AI 8, version 8.0, Thermo Fisher Scientific, Waltham, MA 02451). The respective areas of component peaks were calculated using curve-fitting software (Igor Pro, version 1.6, Wavemetrics, Lake Oswego, OR 97035)³⁸. FTIR frequency limits were: α-helix (1662-1650 cm^-1), β-sheet (1637-1613 cm^-1), turn/bend (1682-1662 cm^-1), and disordered or random (1650-1637 cm^-1)³⁹.

Captive bubble surfactometry

Adsorption and surface tension lowering ability of surfactant preparations were measured with a captive bubble surfactometer at physiological cycling rate, area compression, temperature, and humidity¹⁴. The captive bubble surfactometer used here was a fully-computerized version of that described and built by Schürch and coworkers^40,41. Quasi-static compression and expansion of the air bubble was performed in discrete steps at a rate of 5% of the bubble volume every 10 sec with continuous video recording of the bubble shape. Dynamic compression and expansion cycling was performed between 10 and 110% of the original bubble area at a cycling rate of 20 cycles/min. Both modalities show extreme flattening of the air bubble in active surfactant preparations. We used a B-YL surfactant mixture consisting of 3 mole% of B-YL peptide formulated in surfactant lipids (DPPC:POPC:POPG 5:3:2, mole:mole:mole) with a concentration of 35 mg/mL. Surfactant lipids alone were used as negative control and SMB surfactant (3 mole% of SMB in surfactant lipids) and the clinical surfactant Curosurf® (porcine lung extract containing both SP-B and SP-C and 80 mg/mL of lipids) as positive control. We routinely analyze surfactant preparations at an average surfactant lipid concentration of ~25 μg/mL in the bubble chamber, but as Curosurf® is more concentrated than synthetic surfactant, we applied 1 µL of synthetic surfactant at 35 mg/mL and 0.5 µL of Curosurf® at 80 mg/mL to the bubble chamber (~1.5 mL volume), and performed all measurements in quadruplicate.

In vivo experiments

Animal experiments were performed under established protocols reviewed and approved by the Institutional Animal Care and Use Committee of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center (LA BioMed protocol # 020645). All procedures and anesthesia were in accordance with the American Veterinary Medical Association (AMVA) guidelines. Any suffering of the rabbits was ameliorated by providing optimal anesthesia and sedation as outlined below.

The lung lavage rabbit model represents a relatively pure state of surfactant deficiency over at least 6–8 h and allows for serial measures of arterial blood gases and lung compliance in ventilated, surfactant-deficient animals with a clinical picture of respiratory failure as seen in neonatal RDS and ALI/ARDS. Respiratory failure secondary to surfactant deficiency has a high mortality if not treated with a highly surface-active surfactant preparation. Thirty-three young adult, New Zealand white rabbits, weighing 1.0–1.4 kg, were purchased from IFPS Inc. (Norco, CA). The animals were housed as pairs for a minimum of 24 h in the C.W. Steers Biological Resources Center of LA BioMed, using large cages with non-traumatic and moisture absorbent bedding, and provided with rabbit toys and food and water ad libitum. Husbandry was provided by veterinary technicians under supervision of a veterinarian. The number of animals has been determined from a population correlation=0.6, α=0.05, tails=2, and power=0.8, which gives a sample size of 16 (2x8). Therefore, we generally use 8 animals to test clinical efficacy of an experimental surfactant preparation (here: 3 mole% B-YL in surfactant lipids, n=9) with groups of 8 animals as positive controls (here: Curosurf® and 3 mole% SMB in surfactant lipids, both n=8) and 8 animals for negative controls (here: surfactant lipids alone [DPPC:POPC:POPG 5:3:2 mole:mole:mole], n=8). These treatment group sizes allow significant differences to be found between rabbits receiving an optimal surfactant and positive and negative controls. Animals were assigned to a surfactant preparation using a randomized algorithm and experiments were performed in a special laboratory area set up for the provision of intensive care.

The rabbits received anesthesia with 50 mg/kg of ketamine and 5 mg/kg of acepromazine intramuscularly prior to placement of a venous line via a marginal ear vein. After intravenous administration of 2 mg/kg of propofol and 2 mg/kg of midazolam for anesthesia and sedation, a small incision in the skin of the anterior neck allowed for placement of an endotracheal tube and a carotid arterial line. After insertion of the endotracheal tube, mechanical ventilation was initiated and muscle paralysis induced with intravenous vecuronium (0.1 mg/kg) to prevent spontaneous breathing. During the ensuing duration of mechanical ventilation, anesthesia consisted of continuous intravenous administration of 30 mg/kg/h of propofol and, as needed, additional intravenous dosages of 2 mg/kg of midazolam for sedation, whereas muscle paralysis was maintained with hourly intravenous administration of 0.1 mg/kg of vecuronium. Maintenance fluid was provided with a continuous infusion of lactated Ringer’s solution at a rate of 10 mL/kg/h. Heart rate, arterial blood pressures and rectal temperature were monitored continuously (Labchart® Pro, ADInstruments Inc., Colorado Springs, CO).

The rabbits were ventilated with a volume-controlled rodent ventilator (Harvard Apparatus, South Natick, MA) using a tidal volume 7.5 mL/kg, a positive end-expiratory pressure of 3 cm H₂O, an inspiratory/expiratory ratio of 1:2, 100% oxygen, and a respiratory rate sufficient to maintain the partial pressure of carbon dioxide (PaCO₂) at ^~40 mmHg. Airway flow and pressures and tidal volume were monitored with a pneumotachograph connected to the endotracheal tube (Hans Rudolph Inc., Kansas City, MO). When the partial pressure of oxygen in arterial blood (PaO₂) was >500 mmHg at a peak inspiratory pressure <15 cm H₂O in 100% oxygen, surfactant deficiency was induced with repeated intratracheal instillation and removal of 30 mL/kg of warmed normal saline. When the PaO₂ was stable at <100 mmHg (average 4 lavages), B-YL surfactant or a surfactant control (SMB, Curosurf® or surfactant lipids alone) was then instilled intratracheally at a dose of 100 mg/kg body weight, similar to dosages given to premature infants with RDS. Curosurf® is more concentrated (80 mg/mL) than SMB and B-YL surfactant and lipids alone (35 mg/mL), so Curosurf® was given at 1.25 mL/kg and SMB and B-YL surfactant and lipids alone at 2.9 mL/kg. Oxygenation was followed by measuring arterial pH and blood gases and lung compliance at 15 min intervals over a 2 h period. Dynamic lung compliance was calculated by dividing tidal volume/kg body weight by changes in airway pressure (peak inspiratory pressure minus positive end-expiratory pressure) (mL/kg/cm H₂O).

Animals were sacrificed 2 h after surfactant administration with an overdose (200 mg/kg) of intravenous pentobarbital. End-points were oxygenation and dynamic lung compliance at 120 min after surfactant administration.

Statistical analysis

All data are expressed as mean ± SEM. Statistical analyses (IBM Statistical Package for the Social Sciences (SPSS) 23.0) used Student's t-test for comparisons of discrete data points, and functional data were analyzed by one-way analysis of variance (ANOVA) with Scheffe's post hoc analysis to adjust for multiple comparisons. Differences were considered statistically significant if the P value was <0.05.

Spectroscopic analysis of B-YL and SMB in lipid mimic and surfactant lipid environments

The secondary structures for B-YL in either lipid mimetics (i.e., 40% TFE/60% deuterated-sodium phosphate buffer, pH 7.4 and deuterated aqueous SDS micelles) or surfactant lipids [i.e., deuterated aqueous DPPC: POPC: POPG 5:3:2 (mole:mole:mole) multilayers] were studied with conventional ¹²C-FTIR spectroscopy. Representative FTIR spectra of the amide I band for B-YL in these environments were all similar (Figure 2), each showing a principal component centered at ^~1654–1655 cm^-1 with a small low-field shoulder at ^~1619–1626 cm^-1. Because earlier FTIR investigations of proteins and peptides^39,42 have assigned bands in the range of 1650–1659 cm^-1 as α-helical, while those at ^~1613–1637 cm^-1 are characteristic of β-sheet, B-YL probably assumes α-helical and β-sheet structures and possibly other conformations in these environments. Self-deconvolutions of the Figure 2 spectra confirmed that B-YL is polymorphic, primarily adopting α-helix but with significant contributions from β-sheet, loop-turn and disordered components (Table 1). Interestingly, the relative proportions of secondary conformations determined from FTIR spectra of B-YL (i.e., α-helix > loop-turn ^~ disordered ^~ β-sheet) in both lipid-mimetics and surfactant lipids of varying polarity are all comparable, suggesting an overall stability of the B-YL structure that is remarkably conserved. It is also important to note that the proportions of these secondary conformations are all compatible with B-YL principally assuming an α-helix hairpin^10,14,18.

Figure 2. FTIR spectra of the B-YL mimic in several lipid-mimetics and surfactant lipids.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectral plots show the absorbance (in arbitrary units) as a function of wavenumbers (cm^-1) (See Methods and Results). (A) Deuterated aqueous 40% TFE. (B) Deuterated aqueous SDS. (C) Deuterated aqueous surfactant lipid. In (A), (B) and (C), the IR spectra each show dominant α-helical components centered at 1654–1655 cm^-1 (arrows), with minor bands at ^~1619–1626 cm^-1 (arrows) due to β-sheet, ^~1682–1662 cm^-1 due to loop-turn/bend, and ^~1650–1637 cm^-1 due to disordered or random conformations. Peptide concentrations were 470 μM for the TFE solvent spectra, and 10:1 lipid:peptide (mole:mole) for the SDS detergent and surfactant lipid spectra. The areas under each absorbance curve are normalized.

The secondary conformations for B-YL in lipid mimics (i.e., 40% TFE, aqueous SDS) or synthetic surfactant lipids [aqueous (DPPC:POPC:POPG 5:3:2 mole:mole:mole] were also studied with Circular Dichroism (CD) spectroscopy, to validate the above FTIR results. CD spectra for B-YL in these environments (Figure 3) were all similar, each indicating a major α-helical component characterized by a double minimum at 208 and 222 nm^43–45. Deconvolutions of the Figure 3 spectra showed that B-YL is polymorphic, principally adopting α-helix (^~45–52%), but with significant contributions (i.e., ^~10–26% each) from loop-turn, disordered/random and β-sheet components (Table 1). Interestingly, the secondary conformation proportions determined from CD analysis for B-YL (i.e., α-helix > random ^~loop-turn ^~β-sheet) in both surfactant lipids and lipid mimetics are all compatible with B-YL folding as an α-helix hairpin¹⁸. Moreover, the overall maintenance of these secondary conformations from CD spectra in Table 1 additionally supports our FTIR findings that B-YL assumes a stable 3D-structure in environments of varying polarity.

Figure 3. CD spectra of the B-YL mimic for SP-B in several lipid-mimetics and surfactant lipid.

Circular Dichroism (CD) spectral plots show the mean residue ellipticities ([θ]_MRE x 10^-3 deg-cm² dmol^-1) as a function of wavelength (nm). (A) 40% TFE. (B) SDS. (C) Surfactant Lipid. The double minimum at 208 and 222 nm in each plot indicates that α-helix is the dominant secondary structure for B-YL in these environments. Peptide concentrations were 100 μM. The optical pathlength was 0.01 cm and the temperature was 37°C. Spectra represent the average of 8 scans.

Comparative FTIR spectroscopic studies were next performed on Super Mini-B (SMB) in lipid mimics and surfactant lipids to assess whether the B-YL substitutions in Figure 1 perturb the structure of the parent SMB. The secondary structures for SMB in lipid mimetics (i.e., deuterated 40 % TFE, deuterated aqueous SDS) and surfactant lipids (deuterated aqueous DPPC:POPC:POPG 5:3:2 mole:mole:mole) were investigated with conventional ¹²C-FTIR spectroscopy. Figure 4 shows that representative FTIR spectra of the amide I band for SMB in these environments were similar, each indicating a dominant α-helical component centered at ^~1654–1655 cm^-1 with a small low-field shoulder due to β-sheet at ^~1618–1620 cm^-140,43. Self-deconvolution of the FTIR spectra in Figure 4 demonstrated that SMB in either TFE, SDS or surfactant lipids folds with secondary structures that are characteristic of the α-helix hairpin (Table 1). Our finding that the respective secondary structure profiles for B-YL and SMB are similar in Table 1 suggests that the amino-acid substitutions in B-YL (e.g., four Cys residues replaced by Tyr) do not disrupt the α-helix hairpin conformation.

Figure 4. FTIR spectra of Super Mini-B (SMB) in several lipid-mimetics and surfactant lipids.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectral plots show the absorbance (in arbitrary units) as a function of wavenumbers (cm^-1) (See Methods and Results). (A) Deuterated aqueous 40% TFE. (B) Deuterated aqueous SDS. (C) Deuterated aqueous surfactant lipid. In (A), (B) and (C), the IR spectra each show dominant α-helical components centered at 1654–1655 cm^-1 (arrows), with minor bands at ^~1618–1620 cm^-1 (arrows) due to β-sheet, ^~1682–1662 cm^-1 due to loop-turn/bend, and ^~1650–1637 cm^-1 due to disordered or random conformations. Peptide concentrations were 470 μM for the TFE solvent spectra, and 10:1 lipid:peptide (mole:mole) for the SDS detergent and surfactant lipid spectra. The areas under each absorbance curve are normalized.

Captive bubble surfactometry

Captive bubble surfactometry of B-YL and SMB surfactants (3 mole% peptide in DPPC:POPC:POPG 5:3:2 mole:mole:mole), Curosurf®, and surfactant lipids alone demonstrated excellent surface activity of B-YL (Figure 5). Surface activity of BYL and SMB surfactant and Curosurf® were consistently and equally low during quasi-static cycling with values ≤1 mN/m, indicating that the modifications in the B-YL peptide did not lead to a loss in in vitro surface activity compared to its parent peptide SMB. In contrast, minimum surface tension values of surfactant lipids alone far exceeded those of B-YL, SMB and Curosurf® surfactant and amounted to ^~18 mN/m (p<0.001).

Figure 5. Surface tension reduction activity measured with captive bubble surfactometry.

Surface activity of 3 mole% B-YL and Super Mini-B (SMB) in surfactant lipids were compared with a clinical surfactant (Curosurf®) as positive control and surfactant lipids alone (DPPC:POPC:POPG 5:3:2 mole:mole:mole) as negative control. The lower part of each curve indicates minimum surface tension and the upper part indicates maximum surface tension during 10 compression-expansion cycles. Minimum surface tension values of B-YL and SMB surfactant were similar to those of Curosurf®. Values are mean ± SEM of N=4-5.

In vivo experiments

Animal experiments directly examined the in vivo pulmonary activity of B-YL surfactant when instilled intratracheally in ventilated young adult rabbits with surfactant deficiency and impaired lung function induced by repeated saline lung lavages (Figure 6). Surfactant was administered by intratracheal instillation after the PaO₂ was reduced to stable levels <100 mmHg when breathing 100% oxygen. For this timeframe of study, this model reflects a relatively pure state of surfactant deficiency in animals with mature lungs. Rabbits receiving B-YL, SMB and Curosurf® surfactant had significantly improved arterial oxygenation over the 2 h period of study post-instillation compared to control rabbits instilled with surfactant lipids alone (Figure 6, p<0.001). Dynamic lung compliance also significantly improved over the same period of post-instillation study in rabbits treated with B-YL and SMB surfactants and Curosurf® compared to lipid-only controls (Figure 6). During the first 75 min after surfactant administration, mean PaO₂ values of the B-YL group were slightly lower than in the Curosurf® group (p<0.03), but were not different from those in the SMB group. Thereafter, mean PaO₂ values were similar among the three active surfactant preparations. Dynamic lung compliance of B-YL and SMB surfactants and Curosurf® was not statistically significant different throughout the study period. These data indicate that the modifications in the SP-B peptide mimic B-YL did not lead to a loss in in vivo surface activity, despite a difference in initial kinetics compared to Curosurf®.

Figure 6. Arterial oxygenation and dynamic lung compliance in lavaged, surfactant-deficient, ventilated young adult rabbits treated with surfactant.

Oxygenation (arterial PO₂ in mmHg) and dynamic lung compliance (mL/kg/cm H₂O) of 3 mole% B-YL (n=9) and SMB (n=8) in surfactant lipids were compared with a clinical surfactant (Curosurf®) (n=8) or lipids alone (DPPC:POPC:POPG 5:3:2 mole:mole:mole) (n=8). Surfactant (100 mg/kg) was administered intratracheally as a bolus at time 0. Values are mean ± SEM. Differences in oxygenation and dynamic compliance between B-YL, SMB and Curosurf surfactants were not statistically significant, except for slightly reduced PaO₂ values for B-YL surfactant compared to Curosurf®) (p<0.03), but not to SMB, during the first 75 min post-instillation. Differences between B-YL, SMB and Curosurf®) surfactant with lipids alone were significant (p<0.001).